Bottom Line:
During the ALE experiment, both PB12 and PB13 strains lost the galR and rppH genes, allowing the utilization of an alternative glucose transport system and allowed enhanced mRNA half-life of many genes involved in the glycolytic pathway resulting in an increment in the μ of these derivatives.This is an alternative mechanism to its regeneration from 2-acyl-glycerophosphoethanolamine, whose utilization improved carbon metabolism likely by the elimination of a futile cycle under certain metabolic conditions.The origin and widespread occurrence of a mutated population during the ALE indicates a strong stress condition present in strains lacking PTS and the plasticity of this bacterium that allows it to overcome hostile conditions.

Background: As a metabolic engineering tool, an adaptive laboratory evolution (ALE) experiment was performed to increase the specific growth rate (µ) in an Escherichia coli strain lacking PTS, originally engineered to increase the availability of intracellular phosphoenolpyruvate and redirect to the aromatic biosynthesis pathway. As result, several evolved strains increased their growth fitness on glucose as the only carbon source. Two of these clones isolated at 120 and 200 h during the experiment, increased their μ by 338 and 373 %, respectively, compared to the predecessor PB11 strain. The genome sequence and analysis of the genetic changes of these two strains (PB12 and PB13) allowed for the identification of a novel strategy to enhance carbon utilization to overcome the absence of the major glucose transport system.

Results: Genome sequencing data of evolved strains revealed the deletion of chromosomal region of 10,328 pb and two punctual non-synonymous mutations in the dhaM and glpT genes, which occurred prior to their divergence during the early stages of the evolutionary process. Deleted genes related to increased fitness in the evolved strains are rppH, aas, lplT and galR. Furthermore, the loss of mutH, which was also lost during the deletion event, caused a 200-fold increase in the mutation rate.

Conclusions: During the ALE experiment, both PB12 and PB13 strains lost the galR and rppH genes, allowing the utilization of an alternative glucose transport system and allowed enhanced mRNA half-life of many genes involved in the glycolytic pathway resulting in an increment in the μ of these derivatives. Finally, we demonstrated the deletion of the aas-lplT operon, which codes for the main components of the phosphatidylethanolamine turnover metabolism increased the further fitness and glucose uptake in these evolved strains by stimulating the phospholipid degradation pathway. This is an alternative mechanism to its regeneration from 2-acyl-glycerophosphoethanolamine, whose utilization improved carbon metabolism likely by the elimination of a futile cycle under certain metabolic conditions. The origin and widespread occurrence of a mutated population during the ALE indicates a strong stress condition present in strains lacking PTS and the plasticity of this bacterium that allows it to overcome hostile conditions.

Fig5: Contribution of the deletion of the chromosomal fragment on growth rate in derivatives of the PB11 strain. a The specific growth rates (μ) on glucose as the only carbon source of different PB11 derived strains were determined in order to establish the contribution of some absent genes over the growth in a PTS− strain. b The gene order inside the chromosomal fragment is shown

Mentions:
It has been reported that in strain PB12, the absence of the rppH and galR genes are important for growth rate increases on glucose [2]. To establish the contribution of additional genes within the deleted chromosomal fragment on the growth rate of the evolved strains (Fig. 5b), four different PB11 derived strains were constructed (Fig. 5a). In these derivative strains, rppH (as previously reported for PB12 strain [2]), aas-lplT, or the entire chromosomal fragment were eliminated. The selection of these genes was made after considering their functions and its possible effect on growth (mRNA lifetime for the rppH gene as previously reported, as well as PtdEtn turnover for the aas-lplT operon). A 154 % growth increase was observed in the PB11Δaas-lplT strain, while 261 % and 285 % growth increases were observed in the PB11ΔrppH and PB11Δaas-lplT-rppH double mutant derivatives, respectively, where an epistatic effect was evident (Fig. 5a). Finally, the PB11ΔReg had a similar μ compared to the PB11 double mutant (0.38 h−1 vs 0.37 h−1, respectively), confirming the epistasis observed previously (Fig. 5a). With these results, it is possible to establish that the absence of the rppH gene, the aas-lplT operon, and the galR gene are primarily responsible for the increased μ on glucose in the evolved strains. Other important data from these results imply that the point mutations in the PB12 and PB13 strains have only a minor impact on growth recovery (Fig. 5a). Therefore, the growth increase due to the loss of the chromosomal fragment contributes approximately 86 % in PB12 and 79 % in PB13, while the point mutations in PB12 and PB13 contribute only 14 % and 21 %, respectively.Fig. 5

Fig5: Contribution of the deletion of the chromosomal fragment on growth rate in derivatives of the PB11 strain. a The specific growth rates (μ) on glucose as the only carbon source of different PB11 derived strains were determined in order to establish the contribution of some absent genes over the growth in a PTS− strain. b The gene order inside the chromosomal fragment is shown

Mentions:
It has been reported that in strain PB12, the absence of the rppH and galR genes are important for growth rate increases on glucose [2]. To establish the contribution of additional genes within the deleted chromosomal fragment on the growth rate of the evolved strains (Fig. 5b), four different PB11 derived strains were constructed (Fig. 5a). In these derivative strains, rppH (as previously reported for PB12 strain [2]), aas-lplT, or the entire chromosomal fragment were eliminated. The selection of these genes was made after considering their functions and its possible effect on growth (mRNA lifetime for the rppH gene as previously reported, as well as PtdEtn turnover for the aas-lplT operon). A 154 % growth increase was observed in the PB11Δaas-lplT strain, while 261 % and 285 % growth increases were observed in the PB11ΔrppH and PB11Δaas-lplT-rppH double mutant derivatives, respectively, where an epistatic effect was evident (Fig. 5a). Finally, the PB11ΔReg had a similar μ compared to the PB11 double mutant (0.38 h−1 vs 0.37 h−1, respectively), confirming the epistasis observed previously (Fig. 5a). With these results, it is possible to establish that the absence of the rppH gene, the aas-lplT operon, and the galR gene are primarily responsible for the increased μ on glucose in the evolved strains. Other important data from these results imply that the point mutations in the PB12 and PB13 strains have only a minor impact on growth recovery (Fig. 5a). Therefore, the growth increase due to the loss of the chromosomal fragment contributes approximately 86 % in PB12 and 79 % in PB13, while the point mutations in PB12 and PB13 contribute only 14 % and 21 %, respectively.Fig. 5

Bottom Line:
During the ALE experiment, both PB12 and PB13 strains lost the galR and rppH genes, allowing the utilization of an alternative glucose transport system and allowed enhanced mRNA half-life of many genes involved in the glycolytic pathway resulting in an increment in the μ of these derivatives.This is an alternative mechanism to its regeneration from 2-acyl-glycerophosphoethanolamine, whose utilization improved carbon metabolism likely by the elimination of a futile cycle under certain metabolic conditions.The origin and widespread occurrence of a mutated population during the ALE indicates a strong stress condition present in strains lacking PTS and the plasticity of this bacterium that allows it to overcome hostile conditions.

Background: As a metabolic engineering tool, an adaptive laboratory evolution (ALE) experiment was performed to increase the specific growth rate (µ) in an Escherichia coli strain lacking PTS, originally engineered to increase the availability of intracellular phosphoenolpyruvate and redirect to the aromatic biosynthesis pathway. As result, several evolved strains increased their growth fitness on glucose as the only carbon source. Two of these clones isolated at 120 and 200 h during the experiment, increased their μ by 338 and 373 %, respectively, compared to the predecessor PB11 strain. The genome sequence and analysis of the genetic changes of these two strains (PB12 and PB13) allowed for the identification of a novel strategy to enhance carbon utilization to overcome the absence of the major glucose transport system.

Results: Genome sequencing data of evolved strains revealed the deletion of chromosomal region of 10,328 pb and two punctual non-synonymous mutations in the dhaM and glpT genes, which occurred prior to their divergence during the early stages of the evolutionary process. Deleted genes related to increased fitness in the evolved strains are rppH, aas, lplT and galR. Furthermore, the loss of mutH, which was also lost during the deletion event, caused a 200-fold increase in the mutation rate.

Conclusions: During the ALE experiment, both PB12 and PB13 strains lost the galR and rppH genes, allowing the utilization of an alternative glucose transport system and allowed enhanced mRNA half-life of many genes involved in the glycolytic pathway resulting in an increment in the μ of these derivatives. Finally, we demonstrated the deletion of the aas-lplT operon, which codes for the main components of the phosphatidylethanolamine turnover metabolism increased the further fitness and glucose uptake in these evolved strains by stimulating the phospholipid degradation pathway. This is an alternative mechanism to its regeneration from 2-acyl-glycerophosphoethanolamine, whose utilization improved carbon metabolism likely by the elimination of a futile cycle under certain metabolic conditions. The origin and widespread occurrence of a mutated population during the ALE indicates a strong stress condition present in strains lacking PTS and the plasticity of this bacterium that allows it to overcome hostile conditions.